Magnetic resonance spectroscopy (MRS) has been used for more than two decades to interrogate metabolite distributions in living cells and tissues. Techniques have been developed that allow multiple spectra to be obtained simultaneously with individual volume elements as small as 1 uL of tissue (i.e., 1 x 1 x 1 mm(3)). The most common modern applications of in vivo MRS use endogenous signals from (1)H, (31)P, or (23)Na. Important contributions have also been made using exogenous compounds containing (19)F, (13)C, or (17)O. MRS has been used to investigate cardiac and skeletal muscle energetics, neurobiology, and cancer. This review focuses on the latter applications, with specific reference to the measurement of tissue choline, which has proven to be a tumor biomarker that is significantly affected by anticancer therapies.